| Eucalyptus is one of the most important plantation tree species in southern China.Its fast-growing and high-quality characteristics can effectively alleviate the current contradiction between China’s ecological environment construction and timber safety.Ralstonia solanacearum is one of the most serious diseases currently affecting Eucalyptus.Although the use of pharmaceuticals and biological control methods has achieved certain results,however,disease-resistant breeding is considered to be the fundamental way to solve this disease.Therefore,understanding the molecular mechanism of Eucalyptus in response to bacterial wilt infection can provide theoretical basis and technical support for the targeted cultivation of resistant varieties using molecular biology and genetic engineering methods.Based on the isolation and identification of R.solanacearum in Eucalyptus,this research selected strong pathogenic R.solanacearum to carry out the experiment of Eucalyptus urophylla infection.The uninfected period(0h),pre-infection period(24h after infection)and the xylem tissue of E.urophylla in the later period(4d after infection)was transcriptome sequenced and quantitatively analyzed by metabolome.The differential expression status of transcriptome and metabolome before and after infection with E.urophylla was analyzed.The changes of physiological and biochemical indexes before and after the study are carried out,with a view to analyzing the response mechanism of E.urophylla in response to the infection of R.solanacearum from the level of gene expression and metabolism.The main research results obtained are as follows:(1)Physiological and biochemical index analysis before and after infection by R.solanacearum: Select highly pathogenic strains to infect E.urophylla,and detect the POD,SOD,and PAL vitality values of E.urophylla before and after onset.Compared with the uninfected period(0h),the POD activity value of E.urophylla showed a cumulative upward trend in 4h-7d days after infection;the overall SOD activity value after fluctuations showed a fluctuating trend,and the SOD content decreased first and then increased.The sharp downward trend in 3d can be judged that the infection process of bacterial wilt has a phased stage;the PAL vitality value shows a gradual accumulation and upward trend after infection,and there are multiple fluctuation peaks 48h-7d after infection,which may be related to the body of E.urophylla It is related to the phased metabolic activities when infected by bacterial wilt.(2)Analysis of differentially expressed genes before and after infection by R.solanacearum: Transcriptome sequencing was performed on E.urophylla in uninfected period(0h),pre-infection period(4h after infection)and late stage(4d after infection)A total of 16,018 genes were differentially expressed in the early stage of bacterial wilt(24h)and uninfected disease(0h)of E.urophylla infection,of which 7,706 differentially expressed genes were up-regulated and 8,312 differentially expressed genes were down-regulated;(4d)Compared with uninfected disease(0h),17,472 differentially expressed genes were obtained,of which 8,573 differentially expressed genes were up-regulated and 8,899 differentially expressed genes were down-regulated.There were 8,756 differentially expressed genes in the late susceptible(4d)and early susceptible(24h)eucalyptus xylem tissues,of which 4,564 differentially expressed genes were up-regulated and 4,192 differentially expressed genes were down-regulated.A total of 1,398 differential genes were co-expressed in the three comparison groups.KEGG enrichment analysis showed that the differential genes were mainly enriched in 16 KEGG pathways compared with the early stage of bacterial wilt(24h)and uninfected diseases(0h)of E.urophylla xylem infection.Moderately;differentially expressed genes are significantly enriched in the 10 KEGG pathways at the end of the susceptible stage compared to the non-susceptible stage;at the end of the susceptible stage,the differentially expressed genes are mainly enriched in ubiquitin-mediated proteolysis and carbon metabolism pathways,however,no significant enrichment pathways were found.(3)Identification of differential metabolic substances before and after infection by R.solanacearum: A quantitative analysis of metabolome was performed on E.urophylla infected by R.solanacearum.Of the 130 differential metabolites,89 were significantly upregulated and 41 significantly down-regulated.A total of 114 differential metabolites were screened in the late infection stage and the control,of which 53 were significantly upregulated and 61 were significantly down-regulated.A total of 141 differential metabolites were screened in the early and late stages of bacterial wilt of E.urophylla infection,of which42 were significantly up-regulated and 99 were significantly down-regulated.17 Different metabolites co-exist in the three comparison groups,mainly alkaloids,fatty acids,phenylpropanoids,terpenes and other substances.KEGG significance analysis results showed that the differential metabolites of the comparative group in the pre-infection period and the uninfected period were enriched in 44 pathways;the differential metabolic substances in the comparative group in the late infection and non-infection period were enriched in 34 pathways;The differential metabolites in the comparative group at the late stage of infection were mainly enriched in 33 channels.Among the three comparative groups,the differential metabolites are enriched in phenylalanine,phenylpropane biosynthesis,glucosinolate biosynthesis,and other 19 pathways.It can be speculated that the differential metabolites in these pathways are throughout the tail leaf Eucalyptus responds to the metabolic products of the entire process of bacterial wilt infection.(4)The results of the joint analysis showed that the differential metabolites and differentially expressed genes in the early stage of E.urophylla infection with R.solanacearum were collectively enriched in the Phenylalanine metabolism pathway,valine,leucine and isoleucine In the degradation pathway and 2-Oxocarboxylic acid metabolism pathway;the differential metabolites and differentially expressed genes of E.urophylla in the later stage of bacterial wilt infection are enriched together in cyanogen Cyanoamino acid metabolism pathway,the metabolites involved in the synthesis of phenylpropane are significantly enriched in the metabolome of E.urophylla during the late infection.106 genes from the transcriptome are significantly enriched in the plant hormone signal transduction pathway;each pathway has multiple metabolites and differential gene enrichment,metabolite content and differential gene expression before and after infection There were significant changes in the amount,which may be related to the location and time of the response of E.urophylla after sensing the invasion of pathogens.It also shows that the infection process of R.solanacearum on E.urophylla is a multiple gene and metabolic pathway Complex process. |
